The effect of motion and dynamics on protein function has recently emerged as an important area of research. Recent advances in NMR spectroscopy have allowed the analysis and quantification of motions of individual protein residues. The poliovirus 3C protease, a model for both serine proteases and the piconovirus 3C protease, presents a unique opportunity of study of function and dynamics using NMR spectroscopy and mRNA display because of its relatively small size. Specific Aim I: Characterize the dynamic and basic structural properties of the poliovirus 3C protease, a model for serine proteases, in solution. 2H and 15N relaxation experiments will be utilized to study the dynamic properties of wild-type 3C. This aim will provide information about the functional dynamics and intramolecular communication of the protease. Specific Aim II: Evolve poliovirus 3C protease proteins with enhanced activity using directed evolution. Directed evolution and mRNA display will be used to evolve and select for 3C proteases with higher efficiencies of activity than the wild-type protein. Evolved proteases that exhibit enhanced activity will be screened to identify those with mutations that would not intuitively affect function. Specific Aim III: Map functional connectivities between the evolved sites of 3C protease and the active site. The goal of this aim will be to compare the evolved proteases with the wild-type protein. Dynamic and basic structural changes from the wild-type will be monitored using the identical 2H and 15N relaxation experiments performed in Aim I. Examination of any changes in dynamics could lead to the discovery of cryptic energetic pathways that are important for protein function. The immediate goal of this study is to monitor the dynamic and structural changes in evolved proteases with enhanced activity. Further goals include using directed evolution to select for changes in specificity as well as evolving more stable proteases. Observations from these changes in stability and specificity may elucidate novel drug targets that assist in the eradication of certain diseases. Relevance: The 3C protease is an essential element in the life cycle of picornaviruses which make it an attractive target for antiviral therapy. It is also a model for serine proteases which are proteins that play vital roles in many processes throughout the body. Comparisons made in this study could give new insight into protein design and could also lead to new targets for both pharmaceutical and antiviral therapies that may not have been discovered through conventional means. [unreadable] [unreadable] [unreadable]